Advantages and limitations of brain imaging methods in the research of absence epilepsy in humans and animal models

Journal of Neuroscience Methods 212 (2013) 195–202

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Basic Neuroscience Invited review

Advantages and limitations of brain imaging methods in the research of absence epilepsy in humans and animal models Dmitry N. Lenkov a,1 , Anna B. Volnova c,2 , Anna R.D. Pope b,3 , Vassiliy Tsytsarev d,∗ a

Nevsky Center of Scientific Collaboration, Saint Petersburg, Razjezshaya 43/1 liter A, suite 8N, Saint Petersburg, 192119, Russia Department of General Physiology, St. Petersburg State University, University Embankment 7/9, Saint Petersburg, Russia c Saint Louis University, Department of Experimental Psychology, Saint Louis,3511 Laclede Ave, MO, 63103-2010 United States d Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, United States b

h i g h l i g h t s  The purpose of this review is to analyze possibilities and limitations of some brain imaging methods.  Among imaging methods we described: fMRI, PET, LORETA, SPECT, NIRS, IOS and VSDi.  Samplings of the most relevant data obtained by the above methods are presented.

a r t i c l e

i n f o

Article history: Accepted 25 October 2012 Keywords: Absence epilepsy Brain imaging Epilepsy Near-infrared spectroscopy Imaging methods Optical imaging Photoacoustic Seizures Voltage-sensitive dye

a b s t r a c t The purpose of this review is to analyze research possibilities and limitations of several methods, technical tools and their combinations for elucidation of absence epilepsy mechanisms, particularly the childhood absences. Despite the notable collection of simultaneous recording of clinical electroencephalography (EEG) and behavioral changes in relation to absence seizures, shortcomings of scalp EEG in both spatial resolution and precise detection of subcortical centers have limited the understanding of the fundamental mechanisms of altered brain function during and after recurrent epileptic paroxysms. Therefore, in the past decade, EEG recordings have often been combined with simultaneous imaging methods in epilepsy studies. Among imaging methods, the following ones are used regularly: functional magnetic resonance imaging (fMRI), positron-emission tomography (PET), low-resolution electromagnetic tomography (LORETA), single photon emission spectroscopy (SPECT), near-infrared spectroscopy (NIRS), and optical imaging of intrinsic signals (IOS). In addition, voltage-sensitive dye optical imaging method and even photoacoustic microscopy can be applied to animal models of epilepsy. Samplings of some of the most relevant data obtained by the above methods are presented. It appears that the elaboration of more adequate animal models of the patterns of absence seizures during the early postnatal period is necessary for better correspondence of human and animal absence phenomena. © 2012 Elsevier B.V. All rights reserved.

Contents Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

∗ Corresponding author at: Department of Anatomy and Neurobiology, University of Maryland School of Medicine, HSF II Room S251, 20 Penn Street, Baltimore, MD 21201-1075, United States. Tel.: +1 410 706 8907. E-mail addresses: [email protected] (D.N. Lenkov), [email protected] (A.B. Volnova), [email protected] (A.R.D. Pope), [email protected] (V. Tsytsarev). 1 Tel.: +7 812 717 7808. 2 Tel.: +7 812 652 8434. 3 Tel.: +1 573 201 9175. 0165-0270/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneumeth.2012.10.018

Absence epilepsy (AE) is a specific kind of brief, generalized non-convulsive epileptic seizure that can occur up to ten times per day. These seizures are characterized behaviorally by sudden impairment of consciousness (absence), typically accompanied by bilateral 3–4 Hz spike–wave discharges (SWDs) in EEG (Blumenfeld, 2005; Panayiotopoulos et al., 2008). The onset of this kind of epilepsy is most often childhood in humans and usually has a positive prognosis: after adolescence, seizures of AE cease in more than 60% of patients. This corresponds to the 19th century name of

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AE, petit mal, which reflects the relative softness of the clinical manifestations of AE compared to more severe types of seizures (grand mal). However, it is necessary to explain and explore the danger associated with this type of epilepsy to the family and other people supporting children suffering from AE so that they receive proper care until there is consistent evidence of clear pathological patterns on EEG records. There are many threats to the health and life of children with AE across situations that people may not commonly consider: crossing over roads or railways; using knives, tools, or other sharp objects; and interaction with flammable objects and other dangerous articles or devices. In modern neurology, there are three unique varieties of AE: childhood AE, juvenile AE, and epilepsy with myoclonic absences (Engel, 1996; Panayiotopoulos et al., 2008; Hughes, 2009). Onset of childhood AE is most frequently between 4 and 9 years of age, and 59–73% of cases occur in females (Dura Trave and Yoldi Petri, 2006). Conversely, a number of communications mention the onset of unusually early absence seizures in very young children ranging from 12 to 20 months postnatal evidenced by typical 3–4 Hz SWDs on EEG recordings (Chaix et al., 2003; Giordano et al., 2011; Caraballo et al., 2011). Frequently childhood absence epilepsy has seizures as the only neurological symptom which is not accompanied by either metabolic or other neurological deficits. At the same time, a reduced number of GABA-A receptors in the thalamus accompanies the typical absence seizures (Crunelli et al., 2012). Also there are well known and important roles of thalamus and cortex in mechanisms of generation of absence seizures (Avoli, 2012). Juvenile AE is manifested later, from 9 up to 21 years of age (mean 12.5 years). In many of the above cases, treatments using valproic acid have led to the cessation of paroxysmal SWDs; control inspections of these children 2–7 years later have showed normal EEG patterns and a normal range of cognitive abilities (Shahar et al., 2007). Childhood AE is characterized by brief, usually 5–30 s absence seizures, repeating several times daily. These seizures are characterized by 3–4 Hz bilateral synchronous SWDs on a normal background EEG and the arrest of ongoing behavioral activity including a sudden stopping of movement, a loss or significant impairment of consciousness, and staring eyes (fixation of gaze). These seizure paroxysms have no convulsions, a fact that sets AE apart from other types of epilepsy. Additionally, AE is further differentiated from other types of epilepsy by its sudden onset and termination; there are no evident precursors like “aura” and postictal depression (Panayiotopoulos et al., 2008; Hughes, 2009). The most common and reliable predictor of childhood AE seizures is hyperventilation, seen in up to 100% of cases in some samples (Hughes, 2009; Ma et al., 2009), whereas intermittent photostimulation is predictive of seizures in 56% of juvenile AE patients (Hughes, 2009). Partial preservation of response to sensory stimuli has been noted in patients with AE (Gloor, 1986) and in rats with genetic AE (Drinkenburg et al., 2003). From a neuro-clinical perspective, AE presents a comparatively bright form of idiopathic epilepsy since it is strictly age-dependent and usually has a positive prognosis (Malik et al., 2008). Full recovery from both childhood and juvenile AE occurs in 70–80% of patients, and a decrease in seizure frequency is observed in many of the remaining patients (Hughes, 2009; Wheless et al., 2005). In recent years, drugs for relief of AE have been differentiated into two classes: specific anti-absence drugs (ethosuximide, metsuximide, dimetadion) which induce weak or no influence on patients with others types of epilepsy, and non-specific anti-epileptic drugs with a wider range of effectiveness (lamotrigine, topiramate, valproate, zonisamide, levetiracetam and others) which show effects in both AE and partial/or and generalized seizures contaminated with tonic–clonic convulsions (Manning et al., 2003). Typical drugs

used in the treatment of AE include ethosuximide and its analogs, valproate, and also phenytoin or lamotrigine. In resistant cases of juvenile AE, the aforementioned drugs are combined with carbamazepine and barbiturates, which often prove effective despite the fact that many epileptologysts consider them outdated. EEG recordings in patients with AE are characterized by paroxysmal events. Such recordings show a series of generalized spike–wave discharges (SWDs) with typical frequencies ranging from 3 to 4 Hz. AE seizures are synchronized in both hemispheres of the brain and range in duration from 2–5 s to 15–30 s (Panayiotopoulos et al., 2008; Hughes, 2009). The highest amplitude SWDs usually occur in fronto-medial cortex, with a steady decrease in SWD amplitude both laterally and caudally across the cortex. With some provisos one can say that at least three main theories on the onset and development of absence seizures have been proposed in recent years (Hughes, 2009). First, the “centrencephalic” theory, also known as “thalamic clock” theory, suggests that the seizures originate from a deep-seated diffusely projecting subcortical pacemaker in the midline thalamus. In according to a second theory, “corticoreticular” theory, the cortex seems to play a leading role: spike–wave discharges have a clear focal onset in the neocortex and propagate quickly throughout the cortex. A third, much more recent, theory is based on research that shows that the SWDs from this cortical focus generalize over the cortex and rapidly spread to the thalamus, while thereafter cortex and thalamus drive each other (Meeren et al., 2005). Absence seizures (SWD) are clearly recognizable on EEG (Crunelli and Leresche, 2002; Deleuze et al., 2012). However, the underlying neural mechanisms of such actions are not yet fully understood. It is well known that the ‘spike-and-wave discharge’ is generated by the synchronized activity of cortical and thalamic neurons. GABAergic neurons of the nucleus reticularis play the key role in this process, which accompany the silence of the thalamocortical neurons. Hyperpolarization of these neurons explains why brains are unresponsive to the sensory stimuli during an absence seizure (Crunelli and Leresche, 2002; Leresche et al., 2012). Neither the cortex, nor the thalamus alone can sustain SWDs, though both of them are involved in SWD genesis (Danober et al., 1998; Avoli, 2012). The thalamo-cortical activity is regulated by different monoaminergic and cholinergic connections and controlled by some particular genes. Knockout animals with a deficient of monoaminergic and cholinergic neurons can be very useful for better understanding the genetically based mechanism of the neural circuits involved in the generation of the absence seizures (Crunelli and Leresche, 2002). New imaging techniques have played an important role in the detection of abnormal changes in the brains of patients with epilepsy. These methods include magnetic resonance imaging (MRI), proton magnetic resonance spectroscopy (PMRS), positron emission tomography (PET), diffusion tensor imaging (DTI), and resting functional connectivity functional MRI (fMRI) (Mishra et al., 2011). Unfortunately, epileptogenesis in specifically absence epilepsy has not been as heavily researched using these methods. Nevertheless, research in other forms of epilepsy suggests that the imaging methods may be useful to monitor disease in human and animal models of AE (Chahboune et al., 2009; Mishra et al., 2011). Nuclear magnetic resonance is a physical phenomenon which occurs when the atomic nuclei generate a second oscillating magnetic field, determined by their property which is called spin (Hornak, 2008). Now functional magnetic resonance imaging (fMRI) is the most reliable noninvasive method to detect changes in local blood flow and oxygenation. These changes frequently accompany regional cerebral activation, including epileptic activity. The very common application of fMRI includes the localization of task-correlated language and function (Detre, 2004) and the

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localization of epileptic foci. However, frequent epileptic activity is not accompanied by changes in local blood flow or oxygenation during interictal period. This hinders fMRI application in epileptic research and makes fMRI more useful only in combination with EEG or some other techniques. Positron-emission tomography (PET) is the nuclear imaging, non-invasive technique established in the 1970s. Physically it is based on the detection of the pairs of gamma quants, emitted by a positron-emitting radionuclide, most frequently 2-deoxy-2[18F]fluoro-d-glucose (FDG), which is introduced into the organism just before an imaging session (Casse et al., 2002). As was shown (Casse et al., 2002), epileptogenic sites demonstrated reduced glucose uptake (hypometabolism), but the application of PET in epileptic research is limited because of the high cost, technical difficulties and relatively low spatial and temporal resolution. Nevertheless, development of more specific traces may further improve the clinical value of PET in the epileptic research (Casse et al., 2002). In recent years, the conventional theory about the generalization of absence seizure SWDs to the territory of the neocortex has been called into question. For instance, a combination of two methods, 256 channel EEG scalp recording with equivalent dipole (BESA) and low-resolution electromagnetic tomography (LORETA), has shown a distribution of SWDs onto distal cortical areas both during onset and throughout the duration of absence seizures (Holmes et al., 2004; Tucker et al., 2007; Clemens et al., 2011). In these studies, spikes occurred mostly in ventromedial frontal areas, whereas slow-wave components were limited to frontotemporal areas. In another study using LORETA and interictal EEG in patients with idiopathic generalized epilepsy, an increase in background activity was demonstrated in prefrontal areas; the same territory where SWDs are usually observed in patients with AE (Clemens et al., 2007). Briefly, LORETA is a method for localizing the electric activity in the brain based on multichannel surface EEG recordings, which directly computes a current distribution throughout the brain. This method has been validated in the analysis of epilepsy-related data. This study indicates that the LORETA technique may become a useful method to localize electrical activity in the brain, however, low spatial resolution makes its application limited (Halford, 2003; Lantz et al., 1997; Pascual-Marqui et al., 2002). The earliest imaging studies of AE, by means of positronemission tomography (PET), indicated an increase of neocortical glucose metabolism was found to be related with seizures in children with AE (Ochs et al., 1987; Engel, 1996). In adult patients, similar metabolic changes were not observed. On the other hand, a significant decrease of blood flow that coincides with the onset of seizure paroxysm was revealed in the central cerebral artery in children with AE (Sanada et al., 1988). This decrease in cerebral blood flow during the ictal phase in children with typical absence seizures has been supported recently with the use of a more modern method, single photon emission spectroscopy (SPECT) (Nehlig et al., 2004). SPECT is a gamma ray imaging technique. Technically, it is noninvasive, but as does PET, SPECT requires injection of a gammaemitting tracer into the bloodstream. In many cases, the data obtained by PET and SPECT are similar (Jayalakshmi et al., 2011): ictal SPECT and PET are complementary for localization of the epileptic foci. However, PET and SPECT use different isotopes, so their application is complementary. Both ictal and interictal SPECT are a valuable data for the presurgical evaluation of epileptic patients, but as well as PET, it is limited by technical difficulties, relatively low spatial resolution and high costs. Recently, a fundamental investigation with a group of adult patients diagnosed with hemimegalencephaly whose episodes were always followed by absence paroxysms was carried out using PET and SPECT (Uematsu et al., 2010). A decrease of glucose

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metabolism in the damaged hemisphere of some patients was revealed simultaneously with growth of cerebral blood flow, opposite to the patterns shown in children with AE. Another study, on the other hand, was able to replicate the observation of the intensified glucose metabolism before and during seizures (Bilo et al., 2010). It is necessary to note, that these optical imaging investigations in humans have been carried out with limited samples of patients, which could explain to a degree the aforementioned discrepancy between observations obtained by separate teams of researchers. Simultaneous recording of EEG and Near Infrared Spectroscopy (NIRS) is a technique used to analyze rapid changes in both electrical and cerebral blood flow activities in children and neonates (Roche-Labarbe et al., 2008; Wallois et al., 2010) and in adult humans (Buchheim et al., 2004). NIRS is a method that allows continuous monitoring of tissue oxygenation and hemodynamic changes in the brain not only during interictal period but also during the seizures. As well as fMRI it is based on the monitoring of the local blood flow and oxygenation but it is much cheaper than fMRI, non-invasive, portable and applicable for movable patients and infants. The spatial resolution of NIRS is seriously worse than that same of fMRI. However, some significant results have been obtained by NIRS; it was shown that the convulsive seizures are usually associated with an increase in cerebral blood volume, but absence seizures are associated with a mild decrease in cerebral blood volume of the frontal cortex (Haginoya et al., 2002). NIRS is easily applicable to infants and definitely will provide new information into the pathophysiology of absence epilepsy. The technique has a greater temporal resolution than functional MRI (fMRI) and can measure changes in the tissue concentrations of oxy-, deoxy- and total hemoglobin. In studies used NIRS in with absence seizures, statistically significant difference have been shown between convulsive and absence kinds of epilepsy: an increase of local blood flow in frontal cortical areas during convulsive paroxysm and, oppositely, a decrease of blood flow during AE seizures. Data obtained by the using of 2-photons imaging in vivo, show association of epileptic neural activity with increased capillary blood circulation in a localized cortical area (Hirase et al., 2004). During AE studied in adult patients (Buchheim et al., 2004), significant changes in cerebral Hb-oxygenation were noted during ictal events, there were no pronounced changes in interictal intervals. On the contrary, hemodynamic changes have been observed with NIRS in Sprague–Dawley rats with epileptic spikes induced by injection of bicuculline into sensorimotor cortex (Osharina et al., 2010). Authors demonstrated that these events (HbO and HbT decrease and HbR increase) directly precede the synchronization in electrocorticography (ECoG). The combination of NIRS and video-EEG monitoring shows the usefulness of this noninvasive methodical approach in focus diagnosis of different types epilepsies (Watanabe et al., 2002). NIRS allowed evaluating changes of local levels of hemoglobin and oxygenation and deoxygenating in various brain structures with onset and end of epileptic paroxysm, according to EEG-pattern. It was found that a content of oxyhemoglobin increased insignificantly during first several seconds of seizure activity, after that a decrease of hemoglobin content developed and remained up to the end of paroxysm. Oppositely, a content of deoxyhemoglobin demonstrated little decrease at the onset of paroxysm, and some increase until termination of paroxysm (Roche-Labarbe et al., 2008). Thus, clear advantage of NIRS-imaging has been demonstrated the real possibilities for the introduction of this method into clinical practice. Evidently, however, biological basis of data obtained by means of NIRS-imaging needs some additional fundamental investigations. It emphasizes a potential involvement of astrocytes, raising the question of their roles in the synchronization of neuronal populations. Nevertheless, NIRS combined with video EEG is a promising tool in the diagnosis of epilepsy. After some

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improvements in data acquisition, the combination of it and EEG may become an essential tool in daily clinical routine, particularly in children (Wallois et al., 2010). As was mentioned, 2-photon (or multiphoton) microscopy is an extremely powerful technology which can be applied to many goals including epileptic research (Yang et al., 2005). 2-Photon microscopy is a fluorescence imaging technique that allows 3D imaging of the investigated object up to 1 mm depth in vitro as well as in vivo. 2-Photon microscopy is based on the phenomenon that two or more photons of comparably lower energy than needed for one quantum excitation can simultaneously excite a molecule of the fluorophore. Each excitation long wavelength photon carries part of the energy necessary to excite the fluorophore. An excitation results in the subsequent emission of a photon at a higher energy than either of the excitatory quants. This effect leads to both an increased penetration depth and spatial resolution. 2-Photon is a completely invasive method, moreover, its application for in vivo research is complicated due to brain pulsation, so it is impossible to use it in clinical neuroscience. Nevertheless, 2-photon microscopy remains a powerful technique for a huge spectrum of neuroscience research, including different models of epilepsy. As is well known, astrocytes play important roles in synaptic transmission including pathological neural synchronization during epileptic seizures. 2-Photon microscopy is very well-suited to study neuro-astrocytes physiology in vitro as well as in vivo (Benedikt et al., 2012; Inyushin et al., 2010). Thus, 2-photon imaging was employed to demonstrate abnormal Ca2+ signaling preceding epileptic seizures induced by intracortical injection of 4-aminopyridine (Tian et al., 2006). Also, 2-photon microscopy was employed in the development of non-chemical antiepileptic therapy – focal cooling (Yang et al., 2005). As it was shown by Ca2+ -sensitive 2-photon imaging, focal cooling from 37 to ∼25 ◦ C reduced neurotransmitter release (Yang et al., 2005). Combined EEG recording and fMRI-investigation allows the combination of the high spatial resolution of fMRI with the high temporal resolution of EEG without the low temporal resolution of the former or the low spatial resolution of the later (Gotman et al., 2006; Bai et al., 2010). Therefore, combining of EEG recording and fMRI was used recently in the study of childhood and juvenile AE (Berman et al., 2010; Moeller et al., 2010; Li et al., 2009). Methods of simultaneous EEG–fMRI were used to investigate and compare the BOLD signal changes during interictal and ictal GSWDs in patients with childhood absence epilepsy (Li et al., 2009). In this study authors investigated a homogeneous group of newly diagnosed and untreated children with AE. It was found that both interictal and ictal events are associated with changes of BOLD signal in the basal ganglia–thalamocortical loop. The result of this study showed that thalamic activation is associated with ictal SWD bursts while the activation of cortex areas observed during the interictal SWD. In order to determine the pattern of fMRI changes during typical childhood absence seizures with behavioral impairment, simultaneous EEG–fMRI and behavioral testing has been performed (Berman et al., 2010). Paroxysms in patients with AE were provoked by a hyperventilation, and all seizures could be detected by EEG with continuous observations for patient behavior. The activation in thalamic nuclei have been found during AE seizures in great majority of patients, and deactivation of caudate nucleus and also some neocortical areas; the last deactivation have predeceased to thalamic activation significantly. They found fMRI increases in the “thalamus, frontal cortex, primary visual, auditory, somatosensory, and motor cortex, and fMRI decreases in medial and lateral parietal cortex, cingulate, and basal ganglia during absence seizures”. These findings suggest a cortical–subcortical network, which may be crucial for altered consciousness during absence seizures (Deransart et al., 2003; Paz et al., 2007). In agreement with previous reports (Moeller et al., 2010; Salek-Haddadi et al., 2009), the

investigators found BOLD signal decreases in the basal ganglia (Berman et al., 2010), which has been shown to be important in mechanisms of absence epilepsy in animal models. So, combination EEG–fMRI study during absence seizures may be important for understanding mechanisms of seizure initiation and termination both in clinic and in animal models of AE (Formaggio et al., 2011). The another technique, optical imaging of intrinsic signal (IOS) is based on the principle that action potential firing changes the way neuronal tissue absorbs and scatters light at wavelengths throughout the visible and near-infrared spectrum, including in epilepsy research (Inyushin et al., 2001; Holtkamp et al., 2003; Bahar et al., 2006; Tsytsarev et al., 2008); however, right now IOS is not applicable for absence epilepsy clinical studies. Electrophysiological recording methods, although currently the “gold standard” in mapping epilepsy, are inadequate to address these questions based on restrictions due to volume conduction or sampling limitations. As a low cost, reliable and high resolution method, IOS can be used to image the epileptic foci in clinical practice and animal experiments. The limitation of this technique concerns its applicability to only superficial layers of the brain tissue for imaging due to light absorption of the brain (Schwartz, 2003). Optical recording techniques can overcome many of these limitations by sampling large areas of cortex simultaneously to provide information about blood flow, metabolism and extracellular fluid shifts that are intimately related to excitatory and inhibitory neuronal activity. In fact, optical recordings may actually be more sensitive to certain aspects of epileptic activity than electrophysiological recordings. The IOS has been used to monitor the epileptic seizures in vivo in rats (Chen et al., 2000), ferrets (Schwartz and Bonhoeffer, 2000), non-human primates (Haglund and Hochman, 2007) and humans (Schwartz, 2003). IOS obtains limited application in studies of AE mechanisms until now. It can be explained, partly, by typical generalization of SWDs widely through both hemispheres of the brain in AE patients, whereas IOS possibilities were revealed in studies of strictly localized changes of cortical activity in vivo (Haglund and Hochman, 2007). Another sort of optical imaging, the voltage-sensitive dye imaging (VSD), reflects the neural activity, including epileptic seizures and, therefore, provides information about neural synchronization (Takeshita and Bahar, 2011). It was effectively used for research in antiepileptic drugs and antiepileptic therapy (Haglund, 2012). The method is invasive, and its application in clinical practice is impossible due to the dye toxicity, but in experimental neuroscience it remains a powerful and promising technique. Thus, spiral waves, observed in the neocortex by the voltage-sensitive dye imaging, may contribute to pathological patterns of activity including epileptic seizures (Huang et al., 2010). As well as VSD, photoacoustic imaging (PA), like all other methods of optical imaging, is limited by tissue transparency and scattering (Hu et al., 2009; Liao et al., 2010, 2011, 2012). The spatial resolution is determined by either optical focusing or the ultrasound transducer’s parameters. The spatial resolution depends on imaging depth and can be as high as 1 ␮m, while temporal resolution completely depends on the scanning parameters. At the present time, it is impossible to use PA in clinical brain imaging. Even in animal experiments, it remains completely invasive. However, it can be useful as a powerful tool in the field of cerebrovascular research of epileptic seizures (Liao et al., 2012, 2010; Stein et al., 2009). Furthermore, we ought to mention optical coherence tomography (OCT) which was developed a relatively long time ago, but only recently was used for brain imaging in vivo (Aguirre et al., 2006; Chen et al., 2009). Light scattering in the cortex and induced by this scattering phase shift of optic signal is in the basis of OCT. It provides the highest space resolution with a possibility of

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relatively wide area of brain research. Nevertheless, the recent studies permit to suppose that the alterations of photon dispersion, that induced changes of volume astrocites, may be associated with cortical epileptic activity. This is a possible way for potential application OCT in researches of epileptic phenomena, but until now this method has not been employed for absence epilepsy studies. Addition to imaging methods listed above, that have all been used to locate epileptic foci, there is a new opportunity to indicate epileptic seizures in vivo through usage of the radioactive labeled 2-deoxyglucose (2-DG) analog, fluorescent deoxyglucose substitute, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)amino)-2-deoxyglucose (2-NBDG) in rat model (Tsytsarev et al., 2011a,b, 2012; Yao et al., 2012). Potential radiation exposure limits the usage of the radioactive labeled 2-DG analog. Brain glucose metabolism was subsequently monitored by fluorescence imaging of 2-NBDG. In contrast to 2-DG, 2-NBDG provides optical contrast with low toxicity and fast clearance to urine. Our in vivo results, for the first time, have showed that 2-NBDG is a good indicator of the hypermetabolism caused by epileptic seizures, and may provide a new tool for optically identifying epileptic foci, including the seizures study in model and clinical AE. During last two decades, genetic strains of mutant rodents, particularly rats (WAG/Rij and GAERS) and mice are suitable model objects for investigations of AE mechanisms including metabolic changes. A number of research teams investigate both metabolic and hemodynamic aspects of AE in rats GAERS (Melo et al., 2006; David et al., 2008). In GAERS rats, 5-months-old, AE paroxysms have a duration ca. 20 s and very high frequency of repetition, up to one seizure per minute. Simultaneously with SWD, so-called “freezing” (arrest of movements) is observed usually. The onset of epileptic seizure was followed by some decrease of cortical metabolism, which turned soon to a hyperactivation (Melo et al., 2006). Also, significant enhancement of glucose metabolism was revealed during AE paroxysm in thalamo-cortical system (Dufour et al., 2003; Nehlig et al., 2004). The last finding may be considered as indirect support of a hypothesis about a thalamo-cortical pacemaker, which is responsible for the generation of SWDs (Traub et al., 2005). NIRS-measured oxy- and deoxyhemoglobin in GAERS rats have demonstrated (Roche-Labarbe et al., 2010), that the onset and the end of SWD are correlated with changes in the concentrations of oxy-, deoxy-, and total hemoglobin, that was possibly due to seizure-suppression mechanisms. On rats of another absence strain, WAG/Rij, seizure activity in a form SWDs have been recorded in somatosensory cortex and in some thalamic nuclei earlier than in other parts of the brain (Meeren et al., 2005; Van Luijtelaar and Sitnikova, 2006). In experiments with use of functional MRI (fMRI) of high resolution, a significant increase of blood oxygen-level dependent (BOLD)signal have been observed, mostly bilaterally, in somatosensory and motor cortex, both thalamic and basal nuclei and also in some nuclei of the brain stem of WAG/Rij rats (Nersesyan et al., 2004). The picture of BOLD-activity changes was correlated completely with simultaneously recorded EEG-activity: e.g. in primary visual cortex, which did not involved in seizure paroxysms, none significant shifts in fMRI-activity have been observed. Important conclusion have been resumed: amplitude of BOLD-signal during AE paroxysm reflexes definite extent of involvement of corresponding brain structures into a generation of seizure paroxysm, however the hierarchical interrelations between various brain structures are remained uncertain (Nersesyan et al., 2004; Santisakultarm and Schaffer, 2011). A series of experiments with parallel recording of EEG and fMRI on absence rats of GAERS strain have concluded, that a driver of epileptic activity is localized inside barrel field of somatosensory cortex (David et al., 2008). An activation correlated with paroxysmal seizure at neocortex has been revealed also in thalamic nuclei

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(CM, MD and VL) and in substantia nigra; oppositely, primary visual cortex and supplementary motor area have demonstrated a deactivation during absence seizures (David et al., 2008). The data support findings obtained earlier on absence rats of WAG/Rij strain, concerning most probable localization of primary focus of absence epileptic seizure at barrel field of primary somatosensory cortex and thalamo-cortical loops as a basic physiological substrate for generalization of hypersynchronized SWDs (Meeren et al., 2005; Van Luijtelaar and Sitnikova, 2006; David et al., 2008). Further improvement of MRI led to development “diffusion tensor imaging” (DTI) – technique that allows to visualize the location, the orientation, and the anisotropy of the brain’s white matter tracts on the basis of local microstructural characteristics of water diffusion. Authors (Chahboune et al., 2009; Mishra et al., 2011) employed DTI in rat absence models (GAERS, WAG/Rij) and nonepileptic control strains to detect abnormalities in white matter pathways interconnecting bilateral cortical regions most directly involved in seizure activity. Finding the fractional anisotropy in corpus callosum in young epileptic rats before onset of seizures suggests, that this DTI abnormalities were associated with SWD. Nevertheless, it is not yet clear whether the changes of fractional anisotropy in the anterior corpus callosum were caused by seizures itself or by some other changes accompanying SWD (Mishra et al., 2011). In patients with AE, both ictal and interictal SWDs are associated with BOLD signal changes in the basal ganglia–thalamocortical network (Li et al., 2009), which provides further evidence for basal ganglia-thalamocortical circuit involvement in the generation of SWDs. Also, on absence rats of GAERS strain, it have been shown, that a system of feedback connections between basal nuclei and the neocortex represent a mechanism for online control of absence paroxysms (Deransart et al., 2003; Paz et al., 2007). During an invasion of cortical epileptic discharges into basal nuclei through caudate–thalamic projections, a desynchronization of thalamocortical activity is evoked, which, in one’s turn, brings to a decrease of excitability of epileptized cortical neurons (Fig. 1) and, as a result, to the termination of paroxysm (Paz et al., 2007). Furthermore, a possible role of striato-thalamo-nigral way for transmission of signals controlling absence SWDs have been shown. Anomalous cortical hypersynchronization induces an enhancement of dopaminergic activity of certain striatal neurons during SWD (Deransart et al., 2000). Bilateral microinjections of GABA agonists into subthalamic nuclei, the target of striatal neurons, in absence rats of GAERS strain, brought to an activation of nigro-thalamic neurons in substantia nigra and an increase of inhibitory influences onto thalamic nuclei, and, finally, to antiepileptic effects: SWD’s weakening (Deransart and Depaulis, 2002). Thus, the influence of basal nuclei on SWD during AE may be in control of thalamo-cortical system providing a hypersynchronization of the brain activity, and, in the end, the termination of seizures (Fig. 1). From clinical point of view, striato-nigral system can be considered as a potential target for the application of new antiepileptic drugs (Li et al., 2009), as well as an use some others methods of treatment, e.g. electrical stimulation like the same in Parkinsonism’s patients. Experiments with the use of fMRI in patients have certain difficulties for a realization. Therefore, all not numerous data of such investigations have an important value. In great majority of cases, patients with idiopathic epilepsy including AE have demonstrated none significant morphometric differences. In the study with combined EEG and fMRI (Culver et al., 2003) investigations an activation in left motor cortical area (face and hand representations), as well as in striatum and thalamus have been revealed (Salek-Haddadi et al., 2009). In contrast to experiments on animals, the investigations of human patients are performed usually on small samples, that explains large dispersion of values obtained. However, general

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Fig. 1. Diagram of some cortico-subcortical interconnexions providing onset, development and termination of spike-and-wave discharges (SWDs) during absence epilepsy (rat brain section according to Paxinos and Watson, 2007). Hypersynchronization of excitation of neuronal cluster in deep layers of SI neocortical area provokes an increase of excitatory circulation (red arrows) inside cortico-thalamic system by means of positive feedbacks with some relay thalamic nuclei (Meeren et al., 2005; Polack and Charpier, 2009). Cortical activation of reticular thalamic nucleus (Rt) stimulates burst activity of inhibitory neurons in Rt (black arrow), that leads to synchronization of activity in thalamo-cortical networks and, as a consequence, to maintenance and generalization of SWDs (Pinault, 2003; Timofeev and Steriade, 2004). Mechanism of SWD termination can be explained by an involvement of cortico-basal connexions (Paz et al., 2007): an enhancement of cortical activity provides, presumably, to some increase of inhibition in strio-pallidal system (black arrow from striatum to Globus Pallidus, GP) and to switching off inhibitory input (dotted line) to reticular part of Substantia nigra, SNr. Simultaneously, an increase of excitatory influences (red arrow) onto subthalamic nucleus (Sth) can induce an activation of Sth (Deransart and Depaulis, 2002), which, in one’s turn, brings to hyperexcitation of SNr neurons. Thus, inhibitory influences of SNr onto thalamic relay nuclei significantly increase, and, finally, it can interrupt epileptic paroxysm.

conclusion from the study appears correct: during AE paroxysm, which is identified by EEG recording, metabolic activation is developed at the brain centers involved into thalamo-cortical circuits. Precise determination of primary focus of absence seizures activity is one of most important tasks in studies of AE mechanisms. There is consensus concerning a domination of the focus in absence rats inside deep layers of somatosensory cortex. Numerous data obtained on human patients with AE syndromes do not allow identify a localization of seizure pacemaker in certain area of the neocortex or thalamic nuclei. Additional investigations are necessary in order to decide the problem. Abovementioned results of complex methodical approaches including diverse imaging tools have quite good perspective. In order to approximate experimental animal studies of AE mechanisms nearer to the childhood AE, it appears that forthcoming investigations must be oriented onto elaboration of new models with absence seizures during early postnatal period. Acknowledgment The authors thank Dr. Renee E. Cockerham for help in editing the manuscript. References Aguirre AD, Chen Y, Fujimoto JG, Ruvinskaya L, Devor A, Boas DA. Depth-resolved imaging of functional activation in the rat cerebral cortex using optical coherence tomography. Opt Lett 2006;31(23):3459–61.

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